The plate culture method has been conventionally used for measurement of total live bacterial counts in foodstuffs, biological samples, swab samples, or environmental samples. However, the plate culture method requires time of about two days to one month to obtain a result.
Because of the improvements of sterilization techniques and processing techniques for foodstuffs, needs for distinguishing live states of microorganisms from dead states of microorganisms existing in test samples are increasing even for the cases where the cells exist in an extremely small amount. In the fields of food sanitation inspection and clinical test, in particular, as a quick method for detecting bacteria, it is attempted to determine presence or absence of bacteria or quantify bacteria by amplifying genes specific to the bacteria by PCR to such an amount that the genes can be visually observed. However, if a bacterial DNA is targeted, the background of dead cells originally contained in the test sample is also detected, and therefore a positive result obtained by PCR does not necessarily suggest the presence of live bacteria. Therefore, the current situation in the fields of food sanitation and clinical test is that PCR is not used widely, although it is a highly sensitive and quick technique.
In these days, it is attempted to detect and quantify only live cells of microorganism in a test sample by preparing cDNA with reverse transcriptase for mRNA as a target and performing PCR with primers specific to various bacteria. However, in this method, the reverse transcription of mRNA of dead cells itself is not inhibited, and when 104 cfu/ml or 104 cfu/g or more of dead cells are contained in the test sample, background of the dead cells is detected. Therefore, this method cannot be said to be sufficient as a method for determining the live and dead states.
Specifically, as a method for distinguishing live state from dead states of microorganisms such as bacteria using the PCR method, the methods described in Patent document 1 and 2 have been disclosed. However, the following problems remain in these methods for distinguishing live and dead states of microorganisms such as bacteria using the PCR method.
As for the technique disclosed in Patent document 1, examples are mentioned for distinction of dead cells contained in boiled foodstuffs subjected to high temperature long time sterilization at 100° C. for 10 to 30 minutes, and microorganisms contained in foodstuffs subjected to ethanol sterilization or formaldehyde sterilization. However, especially the treatment of the latter type, there are not foodstuffs actually subjected to such pasteurization treatments. Moreover, there are not supposed detection of only live microorganisms in foodstuffs subjected to the currently major sterilization method in the food industry, low temperature long time pasteurization (LILT pasteurization), high temperature short time pasteurization (HTST pasteurization), or ultra high temperature pasteurization (UHT pasteurization), and detection of only live specific pathogenic bacteria in clinical specimens of infectious disease patients administered with antibiotics. Moreover, in the case of a test sample of a foodstuff or clinical specimen containing dead cells background at a concentration of 104 cfu/ml or higher, the amounts of the final PCR amplified products derived from dead cells exceed the detection limit of the technique of Patent document 1, and therefore it is impossible to determine whether a positive response of a test sample obtained by PCR is derived from live cells or dead cells.
Further, as the technique of Patent document 2, disclosed is a method of distinguishing live cells from dead cells by utilizing relative decrease in RNA/DNA molar ratio of dead cells compared with that of live cells. In this method, the total RNA is extracted, complementary DNA is prepared by using a reverse transcription reaction, then PCR is performed to calculate the Ct value thereof, and the molar concentration of RNA is obtained by using a separately prepared calibration curve. Separately, a region of chromosomal DNA corresponding to that RNA is amplified by PCR to obtain the Ct value thereof, and the molar concentration of the chromosomal DNA is calculated on the basis of the calibration curve to obtain the RNA/DNA molar ratio. That is, the above procedure requires to perform troublesome extraction of total RNA and uses two steps of reverse transcription reaction and PCR. Therefore, this technique is inferior to usual PCR targeting DNA in quantification performance and quickness. Further, RNA is continuously produced in live cells, whereas RNA derived from dead cells is decomposed over time at an early stage. Therefore, the technique lacks stability. Furthermore, in a foodstuff or clinical specimen containing dead cells at a high concentration, only live cells of 1/10 of that concentration can be detected by this technique. Therefore, it is difficult to apply this technique in the fields of food sanitation inspection and clinical test, which require quickness, high sensitivity and accuracy.
A method for selectively detecting live cells (Viable-and-Culturable cells) of a microorganism by distinguishing them from dead cells or injured cells (Viable-but-Non Culturable cells (VNC cells)) is disclosed in Patent document 3. The method disclosed in Patent document 3 is a method comprising the step of treating a test sample with a topoisomerase poison and/or a DNA gyrase poison, the step of extracting DNA from the test sample, and amplifying a target region of the extracted DNA by PCR, and the step of analyzing an amplified product, and as the topoisomerase poison or the DNA gyrase poison, ethidium monoazide is exemplified.
A method in which ethidium monoazide is used is also disclosed in Non-patent document 1. This method is a detection method comprising the step of adding ethidium monoazide to a test sample and irradiating the sample with light, the step of extracting DNA from the sample after the irradiation, and the step of amplifying a specific region by PCR using the extracted DNA as a template. Moreover, a technique of semi-quantitatively quantifying live cells count by a combination of culture of a microorganism and real-time PCR is disclosed in Non-patent document 1.
Moreover, as a method for still more clearly distinguishing live cells and injured cells of a microorganism, the method described in Patent document 4 is disclosed. This method is a method comprising the step of adding a cross-linker capable of cross-linking a DNA by irradiation with light having a wavelength of 350 nm to 700 nm to a test sample, the step of irradiating the test sample to which the cross-linker is added with light having a wavelength of 350 nm to 700 nm, the step of removing the cross-linker contained in the test sample irradiated with light, the step of adding a medium to the test sample from which the cross-linker is removed and incubating the test sample, the step of adding again the cross-linker capable of cross-linking a DNA by irradiation with light having a wavelength of 350 nm to 700 nm to the incubated test sample, the step of irradiating the test sample to which the cross-linker is added with light having a wavelength of 350 nm to 700 nm, the step of extracting a DNA from the test sample and amplifying a target region of the extracted DNA by a nucleic acid amplification method, and the step of analyzing the amplified product.
Meanwhile, there is suggested a possibility that, in amplification of nucleic acid by PCR, albumin may suppress inhibition activity of a PCR inhibitor, or promote the reactions of PCR (Non-patent document 2). Moreover, it is also suggested that calcium inhibits the reactions of PCR, but the inhibition of PCR by calcium can be made tolerable by addition of magnesium ions (Non-patent document 3).
Moreover, there is disclosed a method of performing reactions of PCR using a bacterial DNA as a template, in which the reactions of PCR are performed without extracting the DNA from the bacterium (Non-patent document 4, Patent document 5). In Patent document 5, there is disclosed that random PCR is performed in a bacterium in the DNA fingerprinting method, and phosphates and dodecylsulfates are mentioned as components of the buffer composition for nucleic acid synthesis.